Filter for processing blood and process for producing the same

ABSTRACT

A filter for reducing leukocytes and platelets from an erythrocyte preparation or a whole blood preparation which is characterized by having a filter base coated with a polymer, the content of the polymer being from 0.5 to 10 mg/m 2  per unit area of the total surface of the filter base, and the polymer containing 20% or less of low-molecular weight components having a molecular weight not more than ¼ of the peak top molecular weight in the gel permeation chromatogram.

TECHNICAL FIELD

The present invention relates to a filter for processing blood. Morespecifically, the present invention relates to a filter for reducingleukocytes and platelets from an erythrocyte preparation or a wholeblood preparation at the same time, and to a process for producing thefilter.

BACKGROUND ART

In the field of blood transfusion, in addition to whole bloodtransfusion using a whole blood preparation prepared by adding ananti-coagulating agent to blood collected from a donor, blood componenttransfusion infusing only blood components necessary for the bloodrecipient separated from a whole blood preparation has been commonlypracticed. The blood component transfusion is classified intoerythrocyte transfusion, platelet transfusion, plasma transfusion, andthe like according to the blood component required by the bloodrecipient. As the blood component preparation used for blood componenttransfusion, an erythrocyte preparation, a platelet preparation, a bloodplasma preparation, and the like can be given.

In recent years, leukocyte-free blood transfusion in which leukocytescontained in a blood preparation are removed in advance is applied tothe whole blood transfusion and blood component transfusion. This isbecause of a recent discovery that the leukocytes in the preparationsinduce side effects of blood transfusion such as headache, nausea,chills, anhemolytic exothermic reaction, alloantigen sensitization,GVHD, and virus infection. Therefore, it is necessary to reduceleukocytes in the blood preparation to a level low enough to preventthese side effects.

On the other hand, an erythrocyte preparation may include platelets thatpartly migrate during a centrifuge operation. Since an anhemolytic sideeffect that is suspected to have been caused by platelets has beenreported, it is desirable to reduce platelets to a level as low aspossible. Furthermore, since prion has recently been reported to bepresent in platelets, reduction of platelets not only from anerythrocyte preparation but also from a whole blood preparation isregarded to be highly necessary from the viewpoint of reducing a risk ofprion infection. Therefore, providing a method for reducing plateletsfrom a blood preparation at a high rate is an urgent issue.

As the method for reducing leukocytes from a cell suspension containingleukocytes, a centrifuge method of reducing leukocytes by centrifuge ofthe cell suspension, a filter method of filtering the cell suspensionthrough a filter to cause leukocytes to be adsorbed in the filter, adextran method of adding a physiological saline solution containingdextran to the cell suspension in a blood bag and, after mixing,eliminating a floating leukocyte layer by suction, and the like can begiven. Of these, the filter method is widely accepted due to theadvantages such as excellent leukocyte reducing capability, simpleoperation, and a low cost.

JP 03-158168 A describes that the concentration of leukocytes havingpassed through a fiber laminate exponentially decreases to the thicknessof the fiber laminate. This suggests that, when a cell suspension flowsin the thickness direction of the fiber laminate, leukocytes areadsorbed at a certain probability every time the leukocytes contactcapturing site such as crossing points of the fibers, supporting theabove-described adsorption-elimination theory.

For this reason, investigation for promoting performance of leukocytereducing filters has conventionally been focused on increasing frequencyof contact between leukocytes and fibers, specifically, on decreasingthe average diameter of fibers, increasing the filling density, or usingnonwoven fabrics with a uniform fiber diameter distribution (JP02-203909 A). Quite a few prior art documents have paid attention tochemical properties on the surface of nonwoven fabrics.

Although excellent leukocyte reducing filters and platelet reducingfilters can be obtained by decreasing the average diameter of fibers,increasing the filling density, or using nonwoven fabrics with a uniformfiber diameter distribution, these countermeasures tend to induce biasblood flow, resulting in ineffective performance of the entire filterand fluctuation of leukocyte reduction and platelet reduction.Therefore, improvement of chemical properties on the surface of nonwovenfabrics has been necessary.

Surface modification of nonwoven fabrics by radiation grafting is one ofa few studies dealing with chemical properties on the surface ofnonwoven fabrics (JP 01-249063 A, JP 03-502094 A, etc.). The surface isreformed with an objective of increasing the platelet permeation rate inJP 01-249063 A, whereas the surface is provided with hydrophilicproperties easily to ensure priming with blood in JP 03-502094 A. Thus,neither of the prior art documents has an objective of increasing theadsorption probability of leukocytes, or leukocytes and platelets.

On the other hand, JP 06-247862 A discloses a filter material havingbasic functional groups and nonionic hydrophilic groups at a molar ratioof the basic functional groups to the nonionic hydrophilic groups of notless than 0.6 to less than 6 and having a content of the basicfunctional groups of not less than 5×10⁻⁵ meq/m² to less than 0.1meq/m². However, this filter exhibits an insufficient effect ofsuppressing erythrocyte adhesion, and does not stably ensure theimprovement of the leukocyte reduction performance.

WO 87/05812 discloses a filter element coated with an appropriate amountof a polymer having nonionic hydrophilic groups and basicnitrogen-containing functional groups and containing the basicnitrogen-containing functional groups in an amount of not less than 0.2wt % to less than 4.0 wt %. An example showing superior leukocytereducing capability and platelet permeability of the filter ispresented. WO 87/05812 also presents a comparative example showingincreased reduction of both platelets and leukocytes if a polymercontaining more basic nitrogen-containing functional groups is used. WO87/05812, however, concretely describes the treatment with respect tocattle blood only. No specific information about the treatment withrespect to human blood is given. Performance of reducing leukocytes andplatelets from human blood has not been known. In addition, WO 87/05812does not disclose any information on the reduction of leukocytes andplatelets from an erythrocyte preparation.

The inventors of the present invention have previously prepared a filterfor blood processing by coating a filter substrate with a polymer anddiscovered that the content of low molecular weight components in thepolymer relates to the leukocyte reduction rate in human whole bloodprocessing. The inventors have filed an application for patent on afilter for reducing leukocytes from whole blood (Japanese PatentApplication No. 2000-099715). However, the invention did not investigatesimultaneous reduction of leukocytes and platelets from a whole bloodpreparation or an erythrocyte preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a molecular weight distribution of the polymerused for preparing the filter for blood processing of the presentinvention.

FIG. 2 is a graph showing the relationship between the polymer coatingamount of the filter for blood processing of the present invention andthe reduction rate of platelet from whole blood.

FIG. 3 is a graph showing the relationship between the polymer coatingamount of the filter for blood processing of the present invention andthe reduction rate of platelet from an erythrocyte preparation.

FIG. 4 is an example of X-ray photoelectron spectroscopy (XPS) of thefilter for blood processing of the present invention.

FIG. 5 shows a front elevational view of one embodiment of the devicefor producing the filter for blood processing of the present invention.

DISCLOSURE OF THE INVENTION

In view of this situation, the present inventors have conductedextensive studies to develop a filter for blood processing that canreduce leukocytes and platelets from an erythrocyte preparation or awhole blood preparation at the same time at a high reduction rate.

As a result, the inventors have unexpectedly discovered that a filterfor blood processing having both high leukocyte reducing performance andhigh platelet reducing performance can be obtained if a filter substrateis coated with a specific amount of polymer.

This novel finding has led to the completion of the present invention.

Accordingly, a main object of the present invention is to provide afilter for blood processing that can be effectively used for reducingleukocytes and platelets from an erythrocyte preparation or a wholeblood preparation at the same time and a process for producing thefilter.

The above and other objects, features, and advantages of the presentinvention will become apparent from the following detailed descriptionand the appended claims given in reference to the attached drawings.

The present invention provides a filter for reducing leukocytes andplatelets from an erythrocyte preparation or a whole blood preparation,characterized by having a filter substrate coated with a polymer, thecontent of the polymer being 0.5–10 mg/m² per unit area of the totalsurface of the filter substrate, and the polymer having a molecularweight distribution in which the content of low-molecular weightcomponents having a molecular weight not more than ¼ of the peak topmolecular weight in the gel permeation chromatogram is 20% or less.

The basic features and various preferred embodiment of the presentinvention will now be given for assisting better understanding of thepresent invention.

The present invention is described below in detail.

The filter of the present invention has a filter substrate coated with apolymer. The polymer has a molecular weight distribution in which thecontent of low-molecular weight components having a molecular weight notmore than ¼ of the peak top molecular weight in the gel permeationchromatogram is 20% or less.

The low molecular weight components in the present invention refer tothe components having a low polymerization degree, of which themolecular weight is ¼ or less of the peak top molecular weight, namely,the molecular weight of the component having a maximum strength in thegel permeation chromatogram of the polymer (FIG. 1). Dimers, trimers,oligomers and the like can be given as examples of the low molecularweight components.

In the present invention, the 20% or less content of low-molecularweight components having a molecular weight not more than ¼ of the peaktop molecular weight in the gel permeation chromatogram refers to thatthe proportion of the peak area for the components having a molecularweight not more than ¼ of the peak top molecular weight in the gelpermeation chromatogram shown in FIG. 1 is 20% or less of the total peakarea. In FIG. 1, the horizontal axis represents the molecular weight andvertical axis represents the RI (the intensity measured by adifferential refractive index detector).

The molecular weight and molecular weight distribution of polymers aremeasured by the gel permeation chromatography. Specifically, a solutionof a polymer dissolved in a solution (hereinafter referred to as“Solution A”) prepared by adding LiBr (lithium bromide) toN,N-dimethylformamide to a concentration of 1 mmol is measured at atemperature of 40° C. by an RI (differential refractive index detector)using gel permeation chromatography (GPC)(main body: HLC-8020,manufactured by Tosoh Corp., Japan+analytical program: GPC-LALLS Ver.2.03) connected to a column. The column comprises a guard column (TSKguard column H_(XL)-H, manufactured by Tosoh Corp., Japan) and a maincolumn (a fore column: TSK gel GMH_(XL) B0032, manufactured by TosohCorp., Japan and a rear column: TSK gel α-M B0011, manufactured by TosohCorp., Japan). The measurement is carried out under the condition thatthe solution A is used for the moving phase and the column temperatureis 40° C.

A correlation between a known molecular weight of polymethylmethacrylate (M-M-10 set (manufactured by Polymer Laboratories Inc.,UK)) available from GL Sciences Inc. Japan and a value (retention time)measured by GPC of the polymethyl methacrylate was used to determine themolecular weight and molecular weight distribution of the polymers.

The molecular weight and molecular weight distribution of a polymer maybe determined prior to coating the filter substrate with the polymer. Todetermine molecular weight and molecular weight distribution of apolymer in a filter, the polymer may be extracted from the filter beforedetermination.

The polymer can be extracted from the filter by dipping the filter in asolvent that does not dissolve the filter substrate but dissolves thepolymer. When the polymer is a copolymer of 2-hydroxyethyl(meth)acrylate and dimethylaminoethyl (meth)acrylate,N,N-dimethylformamide and alcohols such as methanol, ethanol, propanolor the like can be used as the solvent. The extracted polymer is driedto remove the solvent and subjected to the molecular weight distributiondetermination according to the above-described method.

The polymer used for preparing the filter of the present invention mustbe refined to reduce low molecular weight components afterpolymerization by a conventional method.

Conventional methods for removing low molecular weight components frompolymers such as a re-precipitation method and fractionation method maybe difficult to reduce the content of low-molecular weight componentshaving a molecular weight not more than ¼ of the peak top molecularweight to 20% or less in the gel permeation chromatogram of the polymer.

The method for reducing the amount of low molecular weight components toobtain a polymer with the above-described molecular weight distributionin the present invention includes, but is not limited to, chromatographymethod, phase separation method or the like. The polymer refining methodby phase separation in the present invention refers to a method ofseparating a polymer solution into a polymer rich layer and a polymerpoor layer by thermally induced phase separation and/or non-solventinduced phase separation and selecting and collecting only the polymerrich layer by fractionation.

If a polymer solution is allowed to stand in a vessel for a prescribedperiod of time after liquid-liquid phase separation, two completelyseparate layers, which a polymer rich layer with high specific gravityis in the lower layer, can be obtained. Therefore the polymer rich layercan be selected and collected by removing the polymer poor layer in theupper layer or by collecting only the polymer rich layer in the lowerlayer.

The liquid-liquid phase separation in the present invention refers to anoperation of separating a polymer solution homogeneously dissolved at acertain temperature into two liquid layers (a polymer rich layer and apolymer poor layer), each layer having a concentration and molecularweight distribution of the polymer different from those of the otherlayer. The liquid-liquid phase separation does not include a phasechange involving deposition of a solid phase or a solid polymer.

The thermally induced phase separation in the present invention refersto a phase separation of a polymer solution homogeneously dissolved at acertain temperature into multiple layers (for example, liquid-liquid,liquid-solid, or liquid-liquid-solid, etc.) by cooling or heating thepolymer solution at a constant rate. Among these, the phase change intoliquid-liquid layers is applied to the present invention.

The non-solvent induced phase separation in the present invention refersto a phase separation of a polymer solution homogeneously dissolved at acertain temperature into multiple layers (for example, liquid-liquid,liquid-solid, or liquid-liquid-solid, etc.) by adding a non-solvent tothe polymer solution. Among these, the phase change into liquid-liquidlayers is applied to the present invention.

When manufacturing a polymer with a particularly high molecular weight,many and various ideas are applied to the manufacturing process toincrease the conversion rate of monomers as high as possible. For thisreason, polymers with a particularly high molecular weight often have anextremely low content of low molecular weight components. When such apolymer with a particularly high molecular weight is used in the presentinvention, the polymer may have the above-described molecular weightdistribution without being subjected to a particular treatment.

A polymer with a weight average molecular weight of 300,000–3,000,000,preferably 300,000–2,000,000, and more preferably 350,000-2,000,000 isused for coating the filter substrate in the present invention. If theweight average molecular weight is less than 300,000, the amount ofmaterials eluted from the filter of the present invention tends toincrease. If the weight average molecular weight is more than 3,000,000,it is difficult for the polymer to be dissolved in a solvent and appliedto the filter substrate.

Any polymer swellable and not dissolved in water may be used in thepresent invention. Examples of such a polymer include, but are notlimited to, polymers having a sulfonic acid group, carboxyl group,carbonyl group, amino group, amide group, cyano group, hydroxyl group,methoxy group, phosphoric acid group, oxyethylene group, imino group,imide group, imino ether group, pyridine group, pyrrolidone group,imidazole group, quaternary ammonium group, and the like, either aloneor in combination.

A copolymer having a nonionic hydrophilic group and a basicnitrogen-containing functional group among these groups is preferable.

A hydroxyl group, amide group, and the like can be given as the nonionichydrophilic group in the present invention.

As examples of the monomer having a nonionic hydrophilic group, monomershaving the above-described hydroxyl group and amide group, such as2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, vinylalcohol (polymerization of vinyl acetate followed by hydrolysis of thepolymer), (meth)acrylamide, and N-vinyl pyrrolidone can be given. Inaddition to the hydroxyl group and amide group, a polyethylene oxidechain can also be given as the nonionic hydrophilic group. As monomershaving a polyethylene oxide chain, alkoxy polyethylene glycol(meth)acrylates such as methoxy ethylene glycol (meth)acrylate, methoxydiethylene glycol (meth)acrylate, methoxy triethylene glycol(meth)acrylate, and methoxy tetraethylene glycol (meth)acrylate can begiven. Among the above monomers, 2-hydroxyethyl (meth)acrylate ispreferably used in view of availability, handling during polymerization,and performance when blood is caused to flow through the filter.

A primary amino group, secondary amino group, tertiary amino group,quaternary ammonium group, and nitrogen-containing aromatic groups suchas a pyridine group and imidazole group can be given as the basicnitrogen-containing functional groups, for example.

As monomers having a basic nitrogen-containing functional group, allylamines; derivatives of (meth)acrylic acid such as dimethylaminoethyl(meth)acrylate, dimethyl aminopropyl (meth)acrylate,3-dimethylamino-2-hydroxyl (meth)acrylate, and the like; styrenederivatives such as p-dimethylaminomethyl styrene, p-dimethylaminoethylstyrene, and the like; vinyl derivatives of a nitrogen-containingaromatic compound such as 2-vinyl pyridine, 4-vinyl pyridine, 4-vinylimidazole, and the like; and derivatives such as quaternary ammoniumsalts prepared by reacting the above vinyl compounds with an alkylhalide or the like can be given. Among the above monomers,dimethylaminoethyl (meth)acrylate and diethylaminoethyl (meth)acrylateare preferably used in view of availability, handling duringpolymerization, and performance when blood is caused to flow through thefilter.

To simultaneously reduce leukocytes and platelets from a whole bloodpreparation or an erythrocyte preparation, the coating amount of thepolymer (mg) for the entire surface area of the filter substrate (m²) isin the range of 0.5–10 mg/m², and preferably 1–7.5 mg/m². If the coatingamount is less than 0.5 mg/m², the leukocyte reduction greatlyfluctuates; if more than 10 mg/m², the platelet reduction may decreaseto a less than 95% level.

Since platelets induce an isoimmunization response, a standard limitingthe number of migrated platelets in erythrocyte preparations to1.5×10¹⁰/unit or less has been established in the Netherlands and othercountries. To sufficiently reduce platelets from a whole bloodpreparation only by filtration without using centrifuge, 95% or higherreduction rate is required.

The entire surface area (m²) in the present invention refers to a valueobtained by multiplying the weight (g) by the specific area (m²/g) ofthe filter substrate to be coated. The entire surface area thus includesnot only the area on the surface of the filter substrate but also thearea in the internal pores of the filter substrate. The specific area inthe present invention refers to a value determined using “Accusorb2100E” (manufactured by Shimadzu Corp., Japan) or an instrument with anequivalent specification. After filling a sample tube with 0.50–0.55 gof the filter substrate and deaerating the tube to 1×10⁻⁴ mmHg (at roomtemperature) for 20 hours, the specific area is determined using kryptongas as an adsorption gas at an adsorption temperature equivalent to aliquid nitrogen temperature.

The coating amount Y (mg/m²) of the polymer for the entire surface area(m²) of the filter substrate of the present invention can be determinedby applying the coating amount X (mg/m²) of the polymer per unit area(m²) of a cut filter substrate, the weight of the substrate per unitarea (Metsuke) A (g/m²), and the specific surface area B (mg/m²) of thesubstrate to the following formula:Y=X/(A×B)

FIG. 2 shows the results of a test conducted by the inventors of thepresent invention for determining the correlation between the coatingamount of the polymer and the platelet reduction performance from wholeblood. In FIG. 2, the rate of platelet reduction from the whole blood isplotted versus the coating amount of the polymer for the entire surfacearea of the filter substrate (g/m²).

The polymer used in this test was a copolymer of 2-hydroxyethyl(meth)acrylate and dimethylaminoethyl (meth)acrylate, and the substratewas a non-woven fabric (Metsuke: 40 g/m², void ratio: 79%, thickness:0.25 mm, specific surface area: 2.01 m²/g) of polyethylene terephthalatefiber with an average fiber diameter of 1.2 μm.

As shown in FIG. 2, the rate of platelet reduction from the whole bloodremarkably increases if the coating amount of the polymer is 10 mg/m² orless.

FIG. 3 shows the results of a test for determining the correlationbetween the coating amount of the polymer and the platelet reductionperformance from an erythrocyte preparation. The same polymer andsubstrate as used in the above platelet reduction performance test fromthe whole blood were used. As shown in FIG. 3, the rate of plateletreduction increases as the coating amount of the polymer decreases. Itcan be seen that the rate of platelet reduction from the erythrocytepreparation also remarkably increases when the coating amount of thepolymer decreases to 10 mg/m² or less.

The specific correlation between the coating amount of the polymer andthe rate of platelet reduction shown in FIGS. 2 and 3 has not been knownin the past, but has been discovered for the first time by the inventorsof the present invention.

The polymer coating ratio for the entire surface of the filter substratein the present invention is preferably less than 70%. The polymercoating ratio (%) is measured by the X-ray photoelectron spectroscopy(XPS) and can be determined from the amount of the polymer in thecovering area of the filter substrate and the amount of the filtersubstrate.

The polymer coating ratio in the in the present invention refers to theproportion of the area coated with the polymer in the entire surfacearea of all the elements composing the filter substrate. Since there isno means for accurately measuring the coating ratio in the entiresurface area of the filter substrate at the present time, the coatingratio in the entire surface area of the filter is measured by XPS andthe resulting value is regarded as a representative value for thecoating ratio. The coating ratio of the polymer in the entire surfacearea of the filter substrate may be less than 70%, and preferably lessthan 50%. This is an important feature of the present invention. Thecoating ratio should be less than 70%, and preferably less than 50%, inorder to achieve the object of the present invention of providing afilter for processing blood possessing both high leukocyte reducingcapability and high platelet reducing capability and capable of beingeffectively used for reducing leukocytes and platelets from erythrocytepreparations and whole blood. If the coating ratio is 70% or more, therate of platelet reduction tends to decrease or greatly fluctuate.

Because the rate of platelet reduction increases as the coating ratiodecreases, the coating ratio is preferably as small as possible in thepresent invention.

The reason for the close relationship between the coating ratio and thefilter performance, particularly the rate of platelet reduction, is notclear, but can be presumed as follows.

Since there are a number of sites on the surface of the filter substrateon which platelets are easily adsorbed (such sites are hereinafterreferred to as “platelet adsorbing sites”), platelets are easilyadsorbed in the filter substrate if caused to contact with the filtersubstrate of which the surface is exposed as large as without beingcoated with the polymer.

The coating ratio of the filter substrate in the filter for bloodprocessing of the present invention can be measured by the X-rayphotoelectron spectroscopy (hereinafter referred to as XPS),conventionally used for measuring chemical conditions on the surface ofan object to a depth of 10 Å to 100 Å, using a monochrome X-ray sourceaccording the following method.

First, an element or a partial structure by which the existence ratio ofthe filter substrate to the polymer is most clearly reflected in the XPSspectrum is selected. This element or partial structure is selectedtaking into consideration the structural difference between the filtersubstrate and the polymer such as an element which is contained in thefilter substrate but not in the polymer and a certain partial structurecommonly possessed by the filter substrate and polymer, but at adifferent ratio.

Then, the XPS spectrums are measured for the standard samples of thefilter substrate and the polymer to determine the ratio of the peak areaof the element or the partial structure selected above to anotherspecific peak area observed in the XPS spectrum. The ratio determinedfor the standard sample of the filter substrate and that of the polymerare respectively designated as X¹ and X².

If the surface of the filter substrate is coated with the polymer, thesurface of the filter is occupied by the filter substrate and thepolymer according to the coating ratio. The existence ratio of thepolymer on the surface of the filter increases as the coating ratioincreases.

As a result, if the XPS spectrum of the filter surface is measured, thespectra for the mixture of the filter substrate sample and the polymersample can be obtained. The ratio X of the peak area for the selectedelement and partial structure to the area of the other specific peakwith respect to this filter is between X¹ and X². This is utilized fordefining the existence ratio (i.e. coating ratio) of the filtersubstrate to the coating material (polymer) on the surface of thefilter.

The method will now be specifically described taking the filter usingpolyethylene terephthalate (hereinafter referred to as “PET”) as thefilter substrate and polyhydroxyethyl methacrylate (hereinafter referredto as “PHEMA”) as the polymer.

Both PET and PHEMA are formed from hydrogen, carbon, and oxygen. Thereis no element contained in either one of the polymers. Therefore, it isimpossible to determine the existence ratio of PET and PHEMA using anelement contained in either one of the polymers as an index. Therefore,the existence ratio of PET to PHEMA is determined taking intoconsideration the content of carbon atom of which the state of chemicalbonding differs in these polymers.

The carbon atoms forming PET and PHEMA can be classified into thefollowing three types:

a) carbonyl carbon atoms,

b) carbon atoms directly bonded to an oxygen atom by a single bond, and

c) carbon atoms not directly bonded to an oxygen atom.

The structural unit forming PET includes two carbonyl carbon atoms, twocarbon atoms directly bonded to an oxygen atom by a single bond, and sixcarbon atoms not directly bonded to an oxygen atom.

The structural unit forming PHEMA includes one carbonyl carbon atom, twocarbon atoms directly bonded to an oxygen atom by a single bond, andthree carbon atoms not directly bonded to an oxygen atom.

The existence ratio (ratio in the number) of the carbon atoms notdirectly bonded to an oxygen atom to carbonyl carbon atoms is 3:1 inboth PET and PHEMA.

The existence ratio (ratio in the number) of the carbon atoms directlybonded to an oxygen atom by a single bond to the carbonyl carbon atomsis 1:1 in PET, whereas that ratio is 2:1 in PHEMA.

The difference of the ratio can be detected as the difference of theratio of the peak intensity (the peak area) in the XPS spectra.

Specifically, the ratio of the peak intensity (area) for the carbonatoms directly bonded to an oxygen atom by a single bond to the peakintensity (area) for the carbonyl carbon atoms is 1:1 in the XPSspectrum of PET, whereas the corresponding ratio in the XPS spectrum ofPHEMA is 2:1.

If the surface of the nonwoven fabric made from PET fiber is coated withPHEMA, the surface of the filter of the present invention is occupied bya mixture of PET and PHEMA according to the coating ratio. The ratio ofthe PHEMA on the surface of the filter increases as the coating ratioincreases. As a result, if the XPS spectrum of the surface of thenonwoven fabric is measured, the spectra of a mixture of PET and PHEMAcan be obtained. The ratio of the peak intensity (area) for the carbonatoms directly bonded to an oxygen atom by a single bond to the peakintensity (area) for the carbonyl carbon atoms is between PET and PHEMA.When the coating ratio is 0%, the ratio of the peak intensity (area) is1:1. The ratio approaches 2:1 as the coating ratio increases and reaches2:1 when the coating ratio becomes 100%.

This is utilized for defining the coating ratio of the filter of thepresent invention obtained by coating the surface of the nonwoven fabricmade from PET fiber with PHEMA.

PET, PHEMA, and the filter of the present invention have peaks at thepositions approximately shown in the XPS spectrum of FIG. 4. In FIG. 4,the peak a is for the carbonyl carbon atoms, the peak b is for thecarbon atoms directly bonded to an oxygen atom by a single bond, and thepeak c is for the carbon atoms not directly bonded to an oxygen atom.

Assuming that the ratio of area for the peak a to the peak b in thestandard PET sample is 1:x, the ratio of areas for the peak a and peak bin the standard PHEMA sample is 1:y, and the ratio of areas for the peaka and peak b in the filter sample is 1:z, the coating ratio of thefilter sample can be defined by the following formula:Coating ratio (%)={|z−x|/|y−x|}×100

The coating ratio of the filter with a combination of filter substrateand polymer other than the combination of PET and PHEMA can be alsodetermined in the same manner.

In the present invention the value of a coating ratio determined for thesurface of a filter in the above-described manner is taken as thecoating ratio of that filter.

It is difficult to obtain a filter with desired performance in thepresent invention unless not only the filter surface but also the entiresurface of the filter substrate elements forming the filter(specifically, the areas inside the filter) is coated with the polymer.Therefore, the following procedure is used to confirm that the filtersubstrate is uniformly coated inside the filter.

First, the filter is cut through an appropriately selected line on thefilter. Next, five points, with respect to each three points of onepoint close to the surface of one side, another point close to thesurface of the other side, and the point at an equivalent distance fromthe two surfaces, are randomly selected on the cross-section surface toobtain XPS spectrum. The configurations of the obtained XPS spectrumshould be confirmed to be equivalent, which means not to besignificantly different from each other. The uniformity of coating inthe thickness direction is evaluated in this manner.

If the filter is cut, the filter substrate is exposed on thecross-section, giving rise to a significant decrease in the apparentcoating ratio on the cross-section. This greatly affects the XPSspectrum and decreases the signal intensity of the polymer in XPSspectrum on the filter cross-section. As a result, the configuration ofXPS spectrum on the filter cross-section becomes different from theconfiguration of XPS spectrum on the filter surface. When comparing theconfigurations of XPS spectra on the cross-section of each filter, themeasuring area must be narrowed, resulting in an increase in noises andmaking it difficult to obtain distinct XPS spectra. Thus, there is nopoint in comparing signal intensity ratios (peak area ratios) of theseXPS spectra. For this reason, the configurations of XPS spectra areregarded to be equivalent when signals are observed at the same point onthe cross-section of each filter, specifically when the chemical shiftsfor signals observed in each XPS spectrum are identical.

The method for preparing the filter for blood processing of the presentinvention will now be described.

The filter for blood processing of the present invention can be preparedby a process comprising (1) coating a filter substrate with a solutionprepared by dissolving a polymer in a solvent (hereinafter referred toas “polymer solution”) or dipping the filter substrate in the polymersolution and (2) removing the excess polymer solution from the filtersubstrate by means of a mechanical compression, gravity, centrifuge, gasblowing, or vacuum suction, or by dipping in a non-solvent to remove thesolvent, followed by drying.

Oxidizing the surface of the filter substrate by γ-ray radiation, UV-rayradiation, corona discharge, or plasma processing, or with chemicalsprior to coating is effective for increasing adhesion of the polymerwith the filter substrate. It is also possible to treat the filtersubstrate and the polymer with heat in a gas or liquid after coating, toincrease adhesion of the polymer with the filter substrate. Acrosslinking reaction in the polymer is also effective to stabilize thecoating layer. The filter substrate may be coated either simultaneouslywith forming the filter substrate or after forming.

As the method for coating the filter substrate with the polymersolution, either a post-measuring method of coating the polymer solutionin an amount larger than the desired coating amount and then reducingthe amount to a prescribed amount or a pre-measuring method ofpreviously measuring a desired coating amount of the polymer solutionand then transferring the solution to the filter substrate can be used.

As the method for drying after coating, a method of drying in dry air, amethod of drying under reduced pressure, a method of drying at roomtemperature, a method of drying with heating, or the like can be used.

Examples of the solvent used for dissolving the polymer, when thepolymer is a copolymer of 2-hydroxyethyl (meth) acrylate anddimethylaminoethyl (meth) acrylate, include glycols such as ethyleneglycol, and diethylene glycol; alcohols such as methanol, ethanol,n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, and t-butylalcohol; N,N-dimethyl formamide, and methyl cellosolve. These solventscan be used either individually or in combination of two or more.Mixtures of these solvents and water may also be used.

As the filter substrate for the filter for blood processing of thepresent invention, in addition to nonwoven fabrics prepared by a meltblow method, flash spinning method, paper milling method, or the like,known filter materials of any form such as paper, woven fabrics, meshfabrics, and porous polymers can be used. Nonwoven fabrics areparticularly preferable. A nonwoven fabric herein refers to a fabricprepared by chemically, thermally, or mechanically combining anaggregation of fibers or threads without weaving or knitting.

As examples of the fiber, synthetic fibers such as polyamide, aromaticpolyamide, polyester, polyacrylonitrile, polytrifluorochloroethylene,polystyrene, polymethyl (meth)acrylate, polyethylene, polypropylene, andpoly-4-methylpentene, and regenerated fibers such as cellulose andcellulose acetate can be given.

The average fiber diameter of the filter substrate of nonwoven or wovenfabrics is 0.3–10 μm, preferably 0.3–3 μm, and more preferably 0.5–1.8μm. If the average fiber diameter is less than 0.3 μm, the pressure losswhen filtering blood may be too large to use the filter in practice; if10 μm or more, on the other hand, leukocytes may not be sufficientlyremoved because leukocytes have a reduced chance of contact with thefiber.

The average filter diameter is measured using an electron microscopephotograph of the sample collected from a woven or nonwoven fabricforming the filter substrate. The average fiber diameter in the presentinvention is determined as follows.

A portion deemed to be substantially homogeneous is sampled from thewoven or nonwoven fabric forming the filter substrate and photographedusing a scanning electron microscope or the like. For sampling, thefilter substrate is divided into squares with one side length of 0.5 cmand six squares are randomly sampled. In random sampling, each dividedsquare is numbered and the required number of squares is selected byusing a table of random numbers, for example. A photograph in thecentral part of each sampled square is taken at a magnification of2,500, from one side (for convenience, hereinafter referred to as Aside) for three earlier sampled squares and another side (forconvenience, hereinafter referred to as B side) for three later sampledsquares. Photographs for the central parts and the neighborhood areas ofeach sampled square are taken until the total number of fibers taken inthe photographs becomes above 100. The diameters of all fibers in thephotographs obtained in this manner are measured. The diameter hereinrefers to the width of fiber in the direction perpendicular to the fiberaxis. Then, the average diameter is determined by dividing the sum ofthe diameters of all measured fibers by the number of the fibers.However, in the case where multiple fibers overlap precluding diametermeasurement of a fiber which hides itself behind another fiber, multiplefibers are consolidated into a fiber with a larger diameter due tofusing or else, or there are fibers with remarkably different diameters,for example, the data obtained are excluded. When the average fiberdiameter on the A side clearly differs from that on the B side of acertain sample, such a sample cannot be regarded as a single filtersubstrate. Here, the term “clearly differs” as used for the averagefiber diameter indicates that the difference is statisticallysignificant. In such an instance, the A side and the B side are regardedas different filter substrates. After identifying the interface of theboth sides, the average fiber diameter for the both sides is separatelymeasured again.

The void ratio of the filter substrate formed from a woven or nonwovenfabric is preferably not less than 50% to less than 95%, and morepreferably not less than 70% to less than 90%. If the void ratio is lessthan 50%, the blood does not flow smoothly; if 95% or more, the filtersubstrate does not have sufficient mechanical strength. The void ratiois determined as follows. The volume (V) of a sample of the filtersubstrate cut to have a prescribed area is determined from the dryweight (W1) and thickness measured for the sample. Then, the sample isdipped in purified water to deaerate and the weight (W2) of the hydratedfilter substrate is measured. The void ratio is then determined applyingthe measured values to the following formula,Void ratio (%)=(W2−W1)×100/ρ/Vwherein ρ indicates the density of purified water.

As examples of the porous polymer, porous polymers made frompolyethylene, polypropylene, poly-4-methylpentene, polyvinylformal,polyacrylonitrile, polysulfone, cellulose, cellulose acetate,polyurethane, polyvinylacetal, polyester, polyamide, polyether imide,poly(meth)acrylate, polyvinylidene fluoride, and polyimide can be given.

The porous polymers have an average pore diameter of 1–60 μm, preferably1–30 μm, and more preferably 1–20 μm. If the average pore diameter isless than 1 μm, a fluid containing leukocytes or platelets such as bloodflows with difficulty; if more than 60 μm, leukocytes may not besufficiently removed because leukocytes have a reduced chance of contactwith the porous polymer. The average pore diameter herein may bemeasured in Porofil liquid (manufactured by Coulter Electronics, Ltd.)using a method conforming to the airflow method described in ASTMF316-86.

The filter for blood processing obtained by the process of the presentinvention can be used by filling the filter in a conventional suitablecontainer for a filter substrate for blood filtration having a bloodoutlet and inlet ports.

In the present invention, either one sheet of the filter may be usedalone or two or more sheets may be used in piles according to thethickness. The number of piled sheets varies according to conditions ofblood filtration. Although the number of sheets is not critical, severalto several tens of sheets are used. When the filter substrate is a wovenor nonwoven fabric, this filter substrate may be used in combinationwith the filter substrates made from other fibers in piles.

To efficiently reduce leukocytes and platelets from an erythrocytepreparation or a whole blood preparation using a filter for bloodprocessing in which two or more sheets of filters are piled, a filter (amain filter) made from a material with the smallest pore size or thesmallest fiber diameter among the filter substrates is preferably coatedwith a polymer having a molecular weight distribution, in which thecontent of low-molecular weight components having a molecular weight notmore than ¼ of the peak top molecular weight in the gel permeationchromatogram is 20% or less. It is more preferable that all the filtersare coated with such a polymer.

In determining the amount of polymer coated on the filter substrateconsisting of combining two or more filter materials with different poresizes or fiber diameters, a single sheet or multiple sheets of thefilter substrate having the same pore size or fiber diameter aremeasured together, then the coated amount is calculated for each filtersubstrate having the same pore size or fiber diameter.

When using two or more sheets of different filter substrates in piles,the coated amount of polymer is 0.5–10 mg/m² for the entire surface ofthe filter substrate. This requires that the filter substrate with thesmallest pore size or fiber diameter (the main filter) be coated withthe polymer in an amount of this range.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described by examples, which should not beconstrued as limiting the present invention.

(Method of Measuring Coating Ratio)

Various kinds of measurement were performed according to the followingprocedure.

1) Measurement of the Polymer Coating Ratio on the Surface of the FilterSubstrate

Samples with a dimension of about 1×1 cm cut from a filter substratewere used for measuring the coating ratio of the polymer on the surfaceof the filter substrate.

Standard film or plate samples made from PET (polyethyleneterephthalate) and standard pellet samples prepared by packing copolymerpowders (copolymer of 2-hydroxyethyl (meth)acrylate anddimethylaminoethyl (meth)acrylate or copolymer of 2-hydroxyethyl(meth)acrylate and diethylaminoethyl (meth)acrylate) were provided. Thesamples were analyzed using an X-ray electronic spectrum (XPS) apparatus(AXIS-Ultra manufactured by Shimadzu Corp., Japan) and an Al Kαmonochrome light source (300 W) for the X-ray source under theconditions of a pass energy by narrow scan of 10 eV and an analysis areaof 700 μm×300 μm, while neutralizing electrostatic charges.

One example of the narrow scan X-ray photoelectron spectrum showing thecorrelation between the intensity (a.u.) (angstrom unit) and bindingenergy (eV) obtained by the above analysis is shown in FIG. 4.

2) Details of Peak Separation

The Eclipse Datasystem Version 2.1 (manufactured by Fisons SurfaceSystem, UK) was used as software peak separation.

Procedure 1: The area for peak separation was specified to include threepeaks a to c shown in the X-ray photoelectron spectrum of FIG. 4 and thebackground was removed by the Shirley method.

Procedure 2: Three Gaussian/Lorentzian mixed peaks corresponding to thepeaks a (originating from the underlined carbon atom in the O—C=O bond),b (originating from the underlined carbon atom in the C—C—O bond), and c(originating from the underlined carbon atom in the C—C—C bond or theC—CH₃ bond) were specified. The center, height, half band width, and theGaussian/Lorentzian mixing ratio of the peaks were used as theparameters for the peak separation.

Procedure 3: The peaks were separated using the minimum Chi-Squaremethod to determine the ratio of the area for the peak a to the area forthe peak b. The following conditions were applied to the peakseparation:

-   -   (1) The difference of the half band width between the peak a and        the peak b of the sample was limited within the corresponding        value of the standard PET sample ±0.1 eV.    -   (2) The Gaussian/Lorentzian mixing ratio for the peaks a and b        was limited to the range of 0.2–0.5, provided that the        Gaussian/Lorentzian mixing ratio for the peak b was (the value        for the peak a) ±0.15. The Gaussian/Lorentzian mixing ratio for        the peak c was limited to the range of 0.2–0.55.

Procedure 4: Assuming that the ratio of areas for the peak a and thepeak b in the standard PET sample is 1:x, the ratio of areas for thepeak a and the peak b in the standard polymer sample (pellets made formthe polymer powder) is 1:y, and the ratio of areas for the peak a andpeak b in the filter sample is 1:z, the coating ratio of the filtersample was determined by the following formula:Coating ratio (%)={|z−x|/|y−x|}×100

Filter samples having a residual solvent content of 1 ppm or less and athickness of 0.1 mm or more were used for the measurement.

3) Measurement of the Coating Ratio Difference in the Thicknessdirection of the Filter Substrate

Narrow Scan: The coating ratio in the thickness direction was measuredin the same manner as in the measurement of the coating ratio on thesurface of the filter substrate described in 1) above, except forapplying the analytical conditions of a pass energy of 40 eV and ananalysis area 27 μmφ were employed.

Five measuring points were randomly selected from a cross-section areanear the surface of one side of the filter, a cross-section area nearthe surface of the other side of the filter, and a cross-section area atan equivalent distance from the two surfaces.

In the following Examples and Comparative Examples, Examples 1-1 to 1-8and Comparative Examples 1-1 to 1-3 relate to processing of erythrocytepreparations, Examples 2-1 to 2-7 and Comparative Examples 2-1 to 2-4relate to processing of whole blood preparations, and Reference Examples1 and 2 relate to a test for eluted materials from polymers.

EXAMPLE 1-1

(Preparation of Polymer Coating Solution)

A copolymer of 97 mol % of 2-hydroxyethyl (meth)acrylate and 3 mol % ofdimethylaminoethyl (meth) acrylate (weight average molecular weight:570,000, basic nitrogen atom content: 0.32 wt %, nonionic hydrophilicgroup content: 97 mol %, basic nitrogen atom: 3 mol %, peak topmolecular weight: 3.75×10⁵, and low molecular weight components: 26.0%)was provided. Ethanol in four times a volume of the polymer solution(copolymer concentration: 39 wt % in ethanol) was added and the polymerwas homogeneously dissolved at 40° C. The solution was allowed to standfor 12 hours at room temperature of 25° C. to separate two liquid phasesby means of thermally induced phase separation. The polymer rich layer(copolymer concentration: 31 wt %) was selected and collected.

The polymer rich layer thus obtained was confirmed to dissolve acopolymer of 2-hydroxyethyl (meth)acrylate and dimethylaminoethyl(meth)acrylate (basic nitrogen atom content: 0.32 wt %, nonionichydrophilic group content: 97 mol %, basic nitrogen atom: 3 mol %, peaktop molecular weight: 3.92×10⁵, and low molecular weight components:14.5%), and to decrease the amount of low molecular weight components inthe polymer, through liquid-liquid phase separation refining.

Ethanol was added to the polymer rich layer to dissolve the polymer at40° C., thereby obtaining a homogeneous polymer solution with acopolymer concentration of 0.06 wt %.

(Coating)

The polymer was coated using the apparatus shown in FIG. 5. Symbols inthe FIG. 5 stand for the following items:

-   -   1: Filter substrate supply roller    -   2: Polymer solution coating vessel    -   3: Thermostat for polymer solution keeping warm    -   4: Roller    -   5: Nip roller    -   6: Roller for rolling up filter    -   7: Filter substrate    -   8: Polymer solution    -   9: Dipping roller    -   10. Water in thermostat    -   11: Drying means (low temperature side)    -   12: Drying means (high temperature side)

A nonwoven fabric (Metsuke: 40 g/m², void ratio: 79%, thickness: 0.25mm, density: 0.16 g/cm³, width: 300 mm, specific area: 2.01 m²/g,length: 30 m), which was made from polyethylene terephthalate with 1.2μm of average fiber diameter, was continuously dipped in the abovesolution at 40° C. using the apparatus shown in FIG. 5 and caused topass through a clearance of 0.13 mm between the rollers. The nonwovenfabric was caused to pass through a first drying chamber at 40° C. (windvelocity: 15 m/sec) for a length of 3 m and then through a second dryingchamber at 60° C. (wind velocity: 15 m/sec) for a length of 3 m,following which the filter was wound around a reel. A fixed line speedof 3 m/min was used. The residual amount of ethanol after passingthrough the first drying chamber was 11%. The residual amount of ethanolafter winding was 1% or less. The filter was efficiently produced withno adhesion among filters after winding. The coating amount on thefilter was 0.62 mg/m² and the coating ratio of the polymer was 20%.

(Evaluation of Blood)

An arbitrarily selected part of the filter thus prepared was cut into anumber of squares, each having a size of 63 mm×63 mm. 36 square sheetswere piled and filled in a container having a blood inlet port and ablood outlet port to a density of about 0.23 g/cm³. The filter had aneffective filtering cross-sectional area of 63 mm×63 mm=3,600 mm² and athickness of 8 mm.

For preparing an erythrocyte preparation, 513 ml of human whole blood(25° C.) consisting of 450 ml of blood and 63 ml of CPD(citrate-phosphate-dextrose) solution was subjected to centrifuge within8 hours after collection to remove platelet-rich plasma and SAGM(saline-adenine-glucose-mannitol) was added as an erythrocytepreservative. 270 ml of a concentrated erythrocyte preparation(hematocrit 60%) thus obtained was used.

The concentrated erythrocyte preparation was passed through the abovefilter at a head drop of 70 cm. The blood processing rate duringfiltration was adjusted to 25 ml/min. Filtration was continued until theblood bag was emptied. The filtered blood was collected.

The leukocyte concentration in the preparation before filtration(prefiltration liquid) and the collected preparation (collected liquid),the volume of the prefiltration liquid, and the volume of the collectedliquid were measured to determine the leukocyte reduction rate accordingto the following formula.

$\begin{matrix}{{{Leukocyte}\mspace{14mu}{reducing}\mspace{14mu}{capability}} = {- {{Log}\lbrack \frac{\begin{matrix}{{leukocyte}\mspace{14mu}{concentration}} \\{{in}\mspace{14mu}{collected}\mspace{14mu}{liquid}}\end{matrix} \times \begin{matrix}{{volume}\mspace{14mu}{of}} \\{{collected}\mspace{14mu}{liquid}}\end{matrix}}{\begin{matrix}{{leukocyte}\mspace{14mu}{concentration}} \\{{in}\mspace{14mu}{prefiltration}\mspace{14mu}{liquid}}\end{matrix} \times \begin{matrix}{{volume}\mspace{14mu}{of}} \\{{prefiltration}\mspace{14mu}{liquid}}\end{matrix}} \rbrack}}} & (1)\end{matrix}$

The volumes of the prefiltration liquid and the collected liquid weredetermined by dividing the respective weight by the specific gravity ofthe blood preparation. The concentrations of leukocytes of theprefiltration liquid and the collected liquid were determined asfollows. A TruCOUNT test tube containing a known number of fluorescencebeads was charged with 600 μl of the sample liquid. 2,400 μl ofLeucoCOUNT reagent was added to the test tube and gently mixed. Themixture was incubated for 5 minutes in a dark place at room temperature.10 TruCOUNT test tubes adjusted in this manner were continuouslymeasured using a flow site meter (FACSCalibur HG manufactured by NipponBecton Dickinson Co., Ltd. Japan). A LeucoCOUNT kit (BD-340523,manufactured by Nippon Becton Dickinson Co., Ltd. Japan) was used as theLeucoCOUNT reagent and TruCOUNT test tubes.

Platelet concentrations in the preparations were measured by amulti-item automated hematology analyzer (K-4500, manufactured by SysmexCorp., Japan) using a stomalizer (manufactured by Sysmex Corp., Japan)as a hemolytic agent. The platelet reduction rate was calculated usingthe following formula.

$\begin{matrix}{{{Platelet}\mspace{14mu}{reduction}\mspace{14mu}{rate}\mspace{11mu}(\%)} = {\quad{\lbrack {1 - \frac{\begin{matrix}{{Platelet}\mspace{14mu}{concentration}} \\{{after}\mspace{14mu}{passing}\mspace{14mu}{through}\mspace{14mu}{the}\mspace{14mu}{filter}}\end{matrix}}{\begin{matrix}{{Platelet}\mspace{14mu}{concentration}} \\{{before}\mspace{14mu}{passing}\mspace{14mu}{through}\mspace{14mu}{the}\mspace{14mu}{filter}}\end{matrix}}} \rbrack \times 100}}} & (2)\end{matrix}$

The blood evaluation test using the filter was repeated three times, andthe results are averaged and shown in Table 1. Leukocyte reducingcapability of 5 Log or more is required for a selective leukocytereducing filter. The results in Table 1 show that the filter has highleukocyte reducing capability.

The filter also showed high platelet reducing capability of a plateletreducing ratio of 95% or more.

EXAMPLE 1-2

An experiment was carried out in the same manner as in Example 1-1,except that a polymer coating solution with a copolymer concentration of1.25 wt % prepared by adding ethanol to the polymer rich layer ofExample 1-1 was used. The coating amount on the filter was 9.55 mg/m²and the coating ratio of the polymer was 50%.

The results of blood evaluation are shown in Table 1. High leukocytereducing capability was obtained. The filter also showed high plateletreducing capability of a platelet reducing rate of 95% or more.

EXAMPLE 1-3

An experiment was carried out in the same manner as in Example 1-1except for using a nonwoven fabric (Metsuke: 40 g/m², void ratio: 75%,thickness: 0.23 mm, density: 0.17 g/cm³, width: 300 mm, specific area:1.98 m²/g) made from poly(trimethyleneterephthalate) fiber with 1.2 μmof average fiber diameter, was used instead of the nonwoven fabric usedin the Example 1-1. The coating amount on the filter was 0.54 mg/m² andthe coating ratio of the polymer was 20%.

The results of blood evaluation are shown in Table 1. High leukocytereducing capability was obtained. The filter also showed high plateletreducing capability of a platelet reduction rate of 95% or more.

EXAMPLE 1-4

An experiment was carried out in the same manner as in Example 1-1except for using a polymer rich layer (copolymer concentration 30 wt %)of a copolymer of 95 mol % of 2-hydroxyethyl (meth) acrylate and 5 mol %of diethylaminoethyl (meth)acrylate (peak top molecular weight:4.08×10⁵, low molecular weight components: 9.9%, basic nitrogen atomcontent: 0.53 wt %, nonionic hydrophilic group content: 95 mol %, basicnitrogen atom: 5 mol %), instead of the polymer rich layer used inExample 1-1. The coating amount on the filter was 0.72 mg/m² and thecoating ratio of the polymer was 20%.

The polymer rich layer of a copolymer of 2-hydroxyethyl (meth)acrylateand diethylaminoethyl (meth)acrylate having a peak top molecular weightof 4.08×10⁵ and a low molecular weight component content of 9.9% wasobtained in the same manner as in Example 1-1 by using a polymersolution (an ethanol solution of copolymer concentration of 41 wt %)containing a copolymer of 95 mol % of 2-hydroxyethyl (meth)acrylate and5 mol % of diethylaminoethyl (meth)acrylate (weight average molecularweight: 650,000, basic nitrogen atom content: 0.53 wt %, nonionichydrophilic group content: 95 mol %, basic nitrogen atom: 5 mol %, peaktop molecular weight: 3.62×10⁵, and low molecular weight components:21.2%). The low molecular weight components in the polymer wereconfirmed to have decreased during purification by the liquid-liquidphase separation.

The results of blood evaluation are shown in Table 1. High leukocytereducing capability was obtained.

The filter also showed high platelet reducing capability of a plateletreduction rate of 95% or more.

EXAMPLE 1-5

An experiment was carried out in the same manner as in Example 1-1except for using a polymer rich layer (copolymer concentration 40 wt %)of a copolymer with the same chemical composition as the polymer ofExample 1-1 (basic nitrogen atom content: 0.32 wt %, nonionichydrophilic group content: 97 mol %, basic nitrogen atom: 3 mol %)having a peak top molecular weight of 3.82×10⁵ and a low molecularweight component content of 12.4%, instead of the polymer rich layerused in Example 1-1. The coating amount on the filter was 0.68 mg/m² andthe coating ratio of the polymer was 20%.

The polymer rich layer of a copolymer of 2-hydroxyethyl (meth)acrylateand dimethylaminoethyl (meth)acrylate having a peak top molecular weightof 3.82×10⁵ and a low molecular weight component content of 12.4% wasobtained by adding a twice volume of ethanol to the polymer solutionbefore purification used in Example 1, homogeneously dissolving thepolymer at 40° C., adding n-hexane in an amount (volume) of 0.033 timethe amount of the polymer solution dropwise while continuing to mixhomogeneously, liquid-liquid phase separating by a non-solvent inducedphase separation method and selecting and collecting only the polymerrich layer. The low molecular weight components in the polymer wereconfirmed to have decreased during purification by the liquid-liquidphase separation.

The results of blood evaluation are shown in Table 1. High leukocytereducing capability was obtained. The filter also showed high plateletreducing capability of a platelet reduction rate of 95% or more.

EXAMPLE 1-6

An experiment was carried out in the same manner as in Example 1-1except for using a polymer rich layer (copolymer concentration: 28 wt %)of a copolymer with the same chemical composition as the polymer ofExample 1-1 (basic nitrogen atom content: 0.32 wt %, nonionichydrophilic group content: 97 mol %, basic nitrogen atom: 3 mol %)having a peak top molecular weight of 3.80×10⁵ and a low molecularweight component content of 18.9%, instead of the polymer rich layerused in Example 1-1. The polymer coating amount on the filter was 0.58mg/m² and the coating ratio of the polymer was 20%.

The polymer rich layer of a copolymer of 2-hydroxyethyl (meth)acrylateand dimethylaminoethyl (meth)acrylate having a peak top molecular weightof 3.80×10⁵ and a low molecular weight component content of 18.9% wasobtained by a combination of thermally induced phase separation andnon-solvent induced phase separation, which comprises adding a thricevolume of purified water to the polymer solution before purificationused in Example 1-1, homogeneously dissolving the polymer at 40° C.,allowing the solution to stand for 12 hours at room temperaturecontrolled to 15° C., and selecting and collecting only the polymer richlayer (copolymer concentration: 28 wt %) by liquid-liquid phaseseparation. The low molecular weight components in the polymer wereconfirmed to have decreased during purification by the liquid-liquidphase separation.

The results of blood evaluation are shown in Table 2. High leukocytereducing capability was obtained. The filter also showed high plateletreducing capability of a platelet reduction rate of 95% or more.

COMPARATIVE EXAMPLE 1-1

An experiment was carried out in the same manner as in Example 1-1except for using a copolymer with the same chemical composition as thepolymer of Example 1-4 (basic nitrogen atom content: 0.53 wt %, nonionichydrophilic group content: 95 mol %, basic nitrogen atom: 5 mol %)having a peak top molecular weight of 3.62×10⁵ and a low molecularweight component content of 20.6%, instead of the polymer rich layerused in Example 1-4.

The results of blood evaluation are shown in Table 2. The polymercoating amount on the filter was 0.50 mg/m² and the coating ratio of thepolymer was 20%.

The copolymer of 2-hydroxyethyl (meth)acrylate and dimethylaminoethyl(meth)acrylate having a peak top molecular weight of 3.62×10⁵ and a lowmolecular weight component content of 20.6% was obtained by dropping thepolymer solution before purification used in Example 1-4 to about a20-fold volume of purified water portion by portion while homogeneouslystirring the solution, re-precipitating the polymer by precipitation,and further drying the precipitate under vacuum at 40° C. The leukocytereducing capability was lower than 5 Log.

COMPARATIVE EXAMPLE 1-2

An experiment was carried out in the same manner as in Example 1-1,except that a polymer coating solution with a copolymer concentration of0.04 wt % prepared by adding ethanol to the polymer rich layer ofExample 1-1 was used.

The results of blood evaluation are shown in Table 2. Leukocyte reducingcapability was evaluated three times to find a significant fluctuationin the leukocyte reducing capability. The average leukocyte reducingcapability was lower than 5 Log. The polymer coating amount on thefilter was 0.40 mg/m² and the coating ratio of the polymer was 20%.

Comparative Example 1-3

An experiment was carried out in the same manner as in Example 1-1,except for using a filter substrate on which a polymer is not coatedinstead of the polymer coated filter used in Example 1-1.

The results of blood evaluation are shown in Table 2. Leukocyte reducingcapability was evaluated three times to find a significant fluctuationin the leukocyte reducing capability. The average leukocyte reducingcapability was lower than 5 Log.

Example 2-1

The experiment was carried out in the same way as in Example 1-1 exceptfor using whole blood for the evaluation.

Specifically, a filter was produced by coating a polymer coat solutionprepared in the same manner as in Example 1-1. In the same manner as inExample 1-1, the content of low molecular weight component was 14.5%,the polymer coating amount on the filter was 0.62 mg/m², and the coatingratio was 20%. 513 ml of human whole blood consisting of 450 ml of bloodand 63 ml of CPD (citrate-phosphate-dextrose) solution was preserved forone day. 270 ml of this blood was filtered and used instead of theerythrocyte preparation. Otherwise, the blood was evaluated in the samemanner as in Example 1-1.

The blood evaluation test using the filter was repeated three times, andthe results are averaged and shown in Table 3. Leukocyte reducingcapability of 5 Log or more was achieved, it was confirmed that thefilter demonstrated high leukocyte reducing capability.

Example 2-2

An experiment was carried out in the same manner as in Example 2-1,except that a polymer coating solution with a copolymer concentration of1.25 wt % prepared by adding ethanol to the polymer rich layer ofExample 2-1 was used. The polymer coating amount on the filter was 8.96mg/m² and the coating ratio of the polymer was 50%.

The results of blood evaluation are shown in Table 3. High leukocytereducing capability and high platelet reduction rate were obtained.

Example 2-3

An experiment was carried out in the same manner as in Example 2-1except for using a nonwoven fabric (Metsuke: 40 g/m², void ratio: 75%,thickness: 0.23 mm, density: 0.17 g/cm³, width: 300 mm, specific area:1.98 m²/g) made from poly(trimethyleneterephthalate) fiber with averagefiber diameter of 1.2 μm was used instead of the nonwoven fabric used inthe Example 2-1. The polymer coating amount on the filter was 0.65 mg/m²and the coating ratio of the polymer was 20%.

The results of blood evaluation are shown in Table 3. High leukocytereducing capability and high platelet reduction rate were obtained.

Example 2-4

An experiment was carried out in the same manner as in Example 2-1except for using a polymer rich layer (copolymer concentration: 29 wt %)of a copolymer of 95 mol % of 2-hydroxyethyl (meth)acrylate and 5 mol %of diethylaminoethyl (meth)acrylate (peak top molecular weight:4.08×10⁵, low molecular weight components: 9.9%, basic nitrogen atomcontent: 0.53 wt %, nonionic hydrophilic group content: 95 mol %, basicnitrogen atom: 5 mol %), instead of the polymer rich layer used inExample 2-1. The polymer coating amount on the filter was 0.60 mg/m² andthe coating ratio of the polymer was 20%.

The results of blood evaluation are shown in Table 3. High leukocytereducing capability and platelet reduction rate were obtained.

Example 2-5

An experiment was carried out in the same manner as in Example 2-1except for using a polymer rich layer (copolymer concentration 40 wt %)of a copolymer with the same chemical composition as the polymer ofExample 1-1 (basic nitrogen atom content: 0.32 wt %, nonionichydrophilic group content: 97 mol %, basic nitrogen atom: 3 mol %)having a peak top molecular weight of 3.82×10⁵ and a low molecularweight component content of 12.4%, instead of the polymer rich layerused in Example 2-1. The polymer coating amount on the filter was 0.68mg/m² and the coating ratio of the polymer was 50%.

The results of blood evaluation are shown in Table 3. High leukocytereducing capability and high platelet reduction rate were obtained.

Example 2-6

An experiment was carried out in the same manner as in Example 2-1except for using a polymer rich layer (copolymer concentration: 28 wt %)of a copolymer with the same chemical composition as the polymer ofExample 1-1 (basic nitrogen atom content: 0.32 wt %, nonionichydrophilic group content: 97 mol %, basic nitrogen atom: 3 mol %)having a peak top molecular weight of 3.80×10⁵ and a low molecularweight component content of 12.9%, instead of the polymer rich layerused in Example 2-1. The polymer coating amount on the filter was 0.62mg/m² and the coating ratio of the polymer was 20%.

The results of blood evaluation are shown in Table 4. High leukocytereducing capability and high platelet reduction rate were obtained.

Example 2-7

An experiment was carried out in the same manner as in Example 2-1,except that a polymer coating solution with a copolymer concentration of0.50 wt % prepared by adding ethanol to the polymer rich layer ofExample 2-1 was used. The polymer coating amount on the filter was 4.58mg/m² and the coating ratio of the polymer was 40%.

The results of blood evaluation are shown in Table 4. High leukocytereducing capability and high platelet reduction rate were obtained.

Comparative Example 2-1

An experiment was carried out in the same manner as in Example 2-1except for using a copolymer with the same chemical composition as thecopolymer of Example 1-1 (basic nitrogen atom content: 0.53 wt %,nonionic hydrophilic group content: 97 mol %, basic nitrogen atom: 5 mol%) having a peak top molecular weight of 3.62×10⁵ and a low molecularweight component content of 20.6%, instead of the polymer rich layerused in Example 2-1. The polymer coating amount on the filter was 0.58mg/m² and the coating ratio of the polymer was 20%.

The results of blood evaluation are shown in Table 4. Blood evaluationwas performed three times to find a significant fluctuation in theplatelet reduction rate. The average platelet reduction rate was lowerthan 95%.

Comparative Example 2-2

An experiment was carried out in the same manner as in Example 2-1,except that a polymer coating solution with a copolymer concentration of0.04 wt % prepared by adding ethanol to the polymer rich layer ofExample 2-1 was used. The results of blood evaluation are shown in Table4. Leukocyte reducing capability was evaluated three times to find asignificant fluctuation in the leukocyte reduction rate. The averageleukocyte reduction rate was lower than 5 Log. The polymer coatingamount on the filter was 0.40 mg/m² and the coating ratio of the polymerwas 20%.

Comparative Example 2-3

An experiment was carried out in the same manner as in Example 2-1,except for using a filter substrate material on which a polymer is notcoated instead of the polymer coated filter used in Example 2-1. Theresults of blood evaluation are shown in Table 4. Leukocyte reducingcapability was evaluated three times to find a significant fluctuationin the leukocyte reducing capability. The average leukocyte reducingcapability was lower than 5 Log.

TABLE 1 Example Example Example Example Example 1-1 1-2 1-3 1-4 1-5Leukocyte reducing 5.0 5.2 5.0 5.1 5.1 capability Platelet reductionrate (%) 99.8 95.8 99.8 99.8 99.9

TABLE 2 Example Comparative Comparative Comparative 1-6 Example 1-1Example 1-2 Example 1-3 Leukocyte 5.0 4.9 4.8 4.2 reducing capabilityPlatelet 99.7 92.0 99.9 98.6 reduction rate (%)

TABLE 3 Example Example Example Example Example 2-1 2-2 2-3 2-4 2-5Leukocyte reducing 5.2 5.4 5.1 5.0 5.1 capability Platelet reductionrate (%) 99.9 95.2 99.9 99.9 99.7

TABLE 4 Example Example Comparative Comparative Comparative 2-6 2-7Example 2-1 Example 2-2 Example 2-3 Leukocyte reducing 5.0 5.3 5.0 4.84.1 capability Platelet reduction 99.0 99.0 93.2 99.6 98.4 rate (%)

Reference Example 1

A filter with a coating in the amount of 73 mg/m² was obtained in thesame manner as in Example 1-4 except for using a polymer coat solutionwith a copolymer concentration of 3 wt %. The filter obtained wasevaluated according to the elution test for rubber materials describedin the standard for sterilized blood transfusion sets (Ministry ofHealth and Welfare, Pharmaceutical and Food Safety Bureau, No. 1079,Dec. 11, 1998). As a result, the filter exhibited potassium permanganatereduction in terms of a consumption difference of 0.21 ml andvolatilization residue of 0.05 mg.

Reference Example 2

A filter with a coating in the amount of 71 mg/m² was obtained in thesame manner as in Reference Example 1 except for using the polymer usedin Comparative Example 1-1. As a result of the same elution test, thefilter exhibited potassium permanganate reduction in terms of aconsumption difference of 0.36 ml and volatilization residue of 0.20 mg.

The results of Reference Examples 1 and 2 show that the filter of thepresent invention is more safe due to a smaller amount of elution.

INDUSTRIAL APPLICABILITY

Using the filter for blood processing of the present invention,leukocytes and platelets causing various side effects after transfusioncan be efficiently and selectively reduced from an erythrocytepreparation or whole blood preparation while preventing a loss of bloodplasma and erythrocytes. The filter for blood processing of the presentinvention can be used with an extreme advantage in manufacturing theblood preparation for pharmaceutical application, medical application,and general industrial application.

1. A filter for reducing leukocytes and platelets from an erythrocytepreparation or a whole blood preparation, comprising a filter substratecoated with a polymer, the content of the polymer being 0.5–10 mg/m² perunit area of the total surface of the filter substrate, and the polymerhaving a molecular weight distribution in which the content ofcomponents having a molecular weight not more than ¼ of the peak topmolecular weight in the gel permeation chromatogram is 20% or less,wherein the polymer coating ratio in the entire surface of the filtersubstrate is less than 70%, wherein the polymer has a weight averagemolecular weight of 300,000–3,000,000, and wherein the polymer isswellable and not dissolved in water.
 2. The filter according to claim1, wherein the polymer has nonionic hydrophilic groups and basicnitrogen-containing functional groups.
 3. The filter according to claim2, wherein the filter substrate is a thermoplastic polymer.
 4. Thefilter according to claim 2, wherein the filter substrate is a nonwovenfabric.
 5. The filter according to claim 1, wherein the filter substrateis a thermoplastic polymer.
 6. The filter according to claim 5, whereinthe filter substrate is a nonwoven fabric.
 7. The filter according toclaim 1, wherein the filter substrate is a nonwoven fabric.
 8. A processfor producing the filter according to claim 1, comprising providing apolymer solution of a polymer mixture which contains unreacted monomersand polymers having a molecular weight not more than ¼ of the peak topmolecular weight in the gel permeation chromatogram in one or moresolvents, separating the polymer solution in a liquid-liquid phase intoa polymer rich layer and a polymer poor layer by thermally induced phaseseparation and/or non-solvent induced phase separation wherein thepolymer rich layer has the polymer having a molecular weightdistribution in which the content of components having a molecularweight not more than ¼ of the peak top molecular weight in the gelpermeation chromatogram is 20% or less, diluting the polymer rich layerselectively collected with a solvent, to form a prepared polymersolution and coating a filter substrate with the prepared polymersolution from the polymer rich layer wherein the polymer coating ratioin the entire surface of the filter substrate is less than 70%, whereinthe polymer has a weight average molecular weight of 300,000–3,000,000,and wherein the polymer is swellable and not dissolved in water.